Transposition Pattern of the Maize Element Ds in Arabidopsis Thaliana

As part of establishing an efficient transposon tagging system in Arabidopsis using the maize elements Ac and Ds, we have analyzed the inheritance and pattern of Ds transposition in four independent Arabidopsis transformants. A low proportion (33%) of plants inheriting the marker used to monitor excision contained a transposed Ds. Selection for the transposed Ds increased this to at least 49%. Overall, 68% of Ds transpositions inherited with the excision marker were to genetically linked sites; however, the distribution of transposed elements varied around the different donor sites. Mapping of transposed Ds elements that were genetically unlinked to the donor site showed that a proportion (3 of 11 tested) integrated into sites which were still physically linked.     

Variable Patterns of Transposition of the Maize Element Activator in Tobacco

The strategy to be followed in a transposon tagging experiment will be determined largely by the transposition pattern of the transposon in question. With a view to utilizing the maize element Activator (Ac) as a transposon tag in heterologous systems, we investigated the pattern of Ac transposition from six different loci in transgenic tobacco. We isolated germinal revertants from plants carrying mutable alleles of the antibiotic-resistant gene streptomycin phosphotransferase (SPT) and mapped the location of the transposed Ac (trAc) elements relative to the donor SPT gene. A comparison of the distributions of trAcs among the six loci revealed that, although the receptor sites for trAcs tend to be linked to the donor locus, the pattern of Ac transposition in tobacco displays surprising locus-to-locus variation. Some trAc distributions showed the same tight clustering around the donor locus previously seen in maize, whereas others were more dispersed. The possible meaning of these findings and their implication for transposon tagging in heterologous systems are discussed.     

Transposition Pattern of the Maize Element Ac from the Bz-M2(ac) Allele

The pattern of transposition of Ac from the mutable allele bz-m2(Ac) has been investigated. Stable (bz-s) and finely spotted (bz-m(F)) exceptions were selected from coarsely spotted bz-m2(Ac) ears. The presence or absence of a transposed Ac (trAc) in the genome was determined and, when present, the location of the trAc was mapped relative to the flanking markers sh and wx. The salient general features of Ac transposition to sites linked to bz are that the receptor sites tend to be clustered on either side of the bz donor site and that transposition is bidirectional and nonpolar. Thus, the symmetrical clustering in the distribution of receptor sites adjacent to bz differs from the asymmetrical clustering reported in 1984 for the P locus by I. M. GREENBLATT. Though Ac tends to transpose preferentially to closely linked sites, an appreciable fraction of Ac transpositions from bz-m2(Ac) is to unlinked sites: 41% among bz-s derivatives and 59% among bz-m(F) derivatives. Many transposition events among the bz-m(F) selections result in kernels carrying a genetically noncorresponding embryo. These can be interpreted as twin sectors arising at one of the megagametophytic mitoses. The bz locus data fit the earlier (1962) model of I. M. GREENBLATT and R. A. BRINK in which transposition takes place from a replicated donor site to either an unreplicated or replicated receptor site.     

Transposition of a Rice Mutator-Like Element in the Yeast Saccharomyces cerevisiae

Mutator-like transposable elements (MULEs) are widespread in plants and are well known for their high transposition activity as well as their ability to duplicate and amplify host gene fragments. Despite their abundance and importance, few active MULEs have been identified. In this study, we demonstrated that a rice (Oryza sativa) MULE, Os3378, is capable of excising and reinserting in yeast (Saccharomyces cerevisiae), suggesting that yeast harbors all the host factors for the transposition of MULEs. The transposition activity induced by the wild-type transposase is low but can be altered by modification of the transposase sequence, including deletion, fusion, and substitution. Particularly, fusion of a fluorescent protein to the transposase enhanced the transposition activity, representing another approach to manipulate transposases. Moreover, we identified a critical region in the transposase where the net charge of the amino acids seems to be important for activity. Finally, transposition efficiency is also influenced by the element and its flanking sequences (i.e., small elements are more competent than their large counterparts). Perfect target site duplication is favorable, but not required, for precise excision. In addition to the potential application in functional genomics, this study provides the foundation for further studies of the transposition mechanism of MULEs.     

The Main Regulatory Region in the Murine PSP Gene is a Parotid Gland Enhancer

The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between –8.0 and –6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between –8.0 to –3.1 kb. This region was shown to activate a heterologous SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.     

The a2m(r-Pa-Pu) Allele of the En-Controlling Element System in Maize

The Enhancer (En) controlling element system in maize includes numerous alleles, one of which, a2m(r-pa-pu), was recovered as a derivative of the a2m(1 1511) allele. The behavior and composition of this allele is described. In the presence of the independently inherited regulatory element, En, the a2m(r-pa-pu) allele mutates and expresses in the aleurone purple, pale and colorless sectors within a colorless background. In the absence of En, the pigmentation is uniformly pale. Colored and colorless germinal mutations of the a2m(r-pa-pu) allele were recovered, although the derivative alleles tested did not respond to En. Uniformly colored derivatives, designated A2', arise only from states of a2m(r-pa-pu) that respond to an active En at relatively early stages in plant development, suggesting that somatic and germinal mutations may arise through similar events. Mutations to A2-expressing alleles appear to occur relatively late in plant development, that is, at or near meiosis, since recognizable mutant sectors do not appear on testcross ears. Kernels exhibiting such mutants are randomly distributed over the ear. The resulting A2' alleles express pigmentation ranging in a continual sequential series from dark pale to intense purple.     

Transposition of Regulatory Elements by P-Element-Mediated Rearrangements in Drosophila melanogaster

Previously we described highly unstable mutations in the yellow locus, induced by the chimeric element and consisting of sequences from a distally located 1A unique genomic region, flanked by identical copies of an internally deleted 1.2-kb P element. Here we show that a sequence, which is part of the yellow 1A region, can be transmitted to the AS-C by successive inversion and reinversion generated by yellow- and AS-C-located P elements. The chimeric element contains a regulatory element from the 1A region that specifically blocks yellow wing and body enhancers and simultaneously stimulates yellow expression in bristles. These results suggest that P-element-generated chimeric elements may play a certain role in rapid changes of regulatory regions of genes during evolution.     

Timing of Transposition of Ac Mobile Element in Potato

The timing of excision of maize transposable element Ac was studied using visual histochemical assay based on Ac excision restoring activity of -glucuronidase (GUS). The Solanum tuberosum L. cv. Bintje was used for Agrobacterium-mediated transformation with pTT230 plasmid harbouring Ac-interrupted gus A gene and npt II gene as a selectable marker gene. Twenty-eight out of 72 kanamycin resistant calli did not express any GUS activity, 31 calli showed partial GUS expression and 13 out of assayed calli revealed strong expression of gus A gene. Plants were regenerated from calli without and/or with partial expression of gus A gene. The regenerated transformants which did not express GUS during the callus phase often contained many small GUS expressing spots on leaves. A phenotypic selection assay for excision of Ac has been also used. This non-detectable excision of Ac in callus tissue could be followed by a "late" timing excision during leaf development. After transformation with pTT224 plasmid harbouring Ac-interrupted hpt II gene and npt II gene transgenic calli containing Ac within the hygromycin resistance gene were derived and hygromycin sensitive plants were regenerated from them. Protoplasts isolated from leaves of transgenic regenerated plants were selected on hygromycin. Hygromycin resistant minicalli showed to harbour multiple copies of Ac and mark out low uniqueness of integration sites.     

A P Element Chimera Containing Captured Genomic Sequences Was Recovered at the Vestigial Locus in Drosophila following Targeted Transposition

A P element carrying the Dopa decarboxylase gene, P[Ddc], was targeted into vg21, a cryptic P element induced mutant allele of the vestigial (vg) locus. The resulting allele, vg28w, contained the expected P[Ddc] plus an additional 9.5 kb of DNA, captured from elsewhere on chromosome II. Reversion of the vg28w mutant allele demonstrated that the entire insert can excise but cannot reinsert at an appreciable frequency. We explain the targeted transposition as the repair of a double stranded gap, created by the excision of the P element at vg21, and suggest that the formation of chimeric elements may be an important component of P element dependent genomic instability.     

Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements

Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.